A Simplified Method for Large Scale Quantification of Transcriptional Activity and Its Use in Studies of Steroids and Steroid Receptors

Abstract

Chloramphenicol acetyltransferase (CAT) is widely used as a reporter to determine the transcriptional specificity of promoters and for the quantification of transcriptional activity of transcription factors such as nuclear receptors. However, large-scale quantification of CAT activity in transfected mammalian cells is still heavily labor-intensive, time-consuming and expensive. Here, we describe a simplified method that combined using multiwell tissue culture plates in transfection and sample preparation and a modified single step method for quantitatively assaying CAT activity. By using multiwell plates, the tedious sample preparation procedure was dramatically simplified. The CAT assay is performed by mixing cell lysate, chloramphenicol, 3H-acetyl co-enzyme A and non-aqueous scintillation fluid in scintillation vials, followed by automatically continuously counting samples two or three cycles at fixed time intervals. The catalytic reaction and determination of CAT activity are carried out in the vials simultaneously. This simplified protocol is faster, less expensive and more accurate than other CAT assay procedures and the results can be normalized easily. The utility of the assay is demonstrated by the analysis of the transcriptional activity of the glucocorticoid and androgen receptors cotransfected into cells with a CAT reporter.

Evidence Denies the Presence of O-GlcNAcylation on Mouse Glucocorticoid Receptor and Its Potential Involvement in Receptor Transcriptional Activity

“…pBAG, which was used as transfection and expression control, contains LacZ gene (galactosidase) whose expression is under the control of Moloney murine leukemia virus long terminal repeat (29). The vectors were transfected into Cos-7 cells by calcium-phosphate precipitation method as described previously (24,30). Transcriptional activity of the GR was induced by treatment of the transfected cells with steroids for 48 hr starting 18 hr after the transfection.…”

“…Transcriptional activity of the GR was induced by treatment of the transfected cells with steroids for 48 hr starting 18 hr after the transfection. The activity in the cells was determined by the CAT assay following the protocol described in detail in our previous publication (24,30).…”